Purification and use of gellan in electrophoresis gels

ABSTRACT

Gellan can be purified from nucleic acid contamination by combining the contaminated gellan with DNase under conditions that allow the DNase to degrade the nucleic acid contaminant. The purified gellan is useful in gel electrophoresis. A buffer which allows cystamine to be used as a reversible cross-linker does not have to be recirculated during the course of a normal gel run.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/427,988 filed Nov. 20, 2002, where this provisionalapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to gellan purification, electrophoresisgel compositions containing purified gellan, and methods relatedthereto.

2. Description of the Related Art

Gel electrophoresis is currently employed for the separation of chargedbiological macromolecules such as proteins and nucleic acids. In gelelectrophoresis, a mixture of charged species is resolved into itscomponents owing to different mobilities of these species in a gelmedium under an imposed electric field. The mobilities depend in largepart on the characteristics of the charged species, including their netsurface charge, which is affected by molecular size and shape.

Many types of gel material are suitable for use as the electrophoresismedium. The gel is often the determining factor in achieving asuccessful resolution of biological macromolecules, and accordingly thedevelopment of suitable gel materials has been the subject of intenseresearch. Many gels are commercially available, and are typicallycomposed of natural or synthetic polymers. Agarose is the most widelyused natural material and polyacrylamide gels are the most commonsynthetic matrix.

In recent years, reversible gels have become commercially available andare increasingly popular for the purpose of preparative applications. Inpreparative applications, sections of the gel medium containing targetbiomolecules are reverted to the solution phase and the biomoleculestherein are recovered by various means. For example, U.S. Pat. No.5,143,646 describes the use of polysaccharide gel blends for stackingelectrophoresis systems, wherein the gels are described as being“thermoreversible” and “pH reversible”. In these particular reversiblegels, the structure of the gel matrix is converted to a liquid form whenthe gel is subjected to heat or pH variation. Concerns have been raised,however, with regard to the conditions for reverting the“thermoreversible” or “pH reversible” gels to solutions.Problematically, the high temperature or specific pH (lower than 3 orhigher than 9) needed for liquefying the gels can denature or otherwisealter the biomolecules contained in the gel matrix.

U.S. Pat. No. 6,203,680 discloses that gellan gum may be used as areversible electrophoresis gel medium. Gellan-based gels have theadvantage of being reversible under relatively mild conditions, andtherefore address the concern about having the liquefication conditionsharm the biomolecules. Gellan gum can be liquefied under mild conditionsbecause it can form a cross-linked gel in the presence of eitherdivalent cations or diamines. In the case where divalent cations areused as the cross-linking agent, liquefaction of the gel may be achievedby adding a sequesting agent specific for the divalent cation. Whendiamine is used as the cross-linking agent, the pH of the gel ismaintained at a value such that the amino groups of the diamine areprotonated. The gellan gel reverts to a liquid solution when the gel pHis adjusted so that the amino groups of the cross-linking agent are nolonger protonated. This can be achieved under relatively mild pHconditions. Gellan-based electrophoresis gel can also be formed in thepresence of cross-linked diamines that contain disulfide bonds. Gelformed in this way can be returned to solution using a reducing agent tobreak the disulfide bonds. A typical disulfide-containing cross-linkeris cysteine dimethyl ester, also referred to as cystamine.

Gellan gum can be purified via a series of deionization andprecipitation steps as described in Doner et al., “Purification ofCommercial Gellan to Monovalent Cation Salts Results in AcuteModification of Solution and Gel-Forming Properties,” CarbohydrateResearch (1995), 273, 225-233. This purification procedure is timeconsuming and costly. Therefore, there exists a need in the art forgellan purified in an alternative and inexpensive way so it becomeseconomical to use gellan as a replacement for agarose.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method by which gellan, includingcommercial preparations of gellan, can be modified to render themparticularly useful in electrophoresis gels. For instance, the presentinvention provides a method for purifying gellan where the methodincludes: (a) combining DNase and gellan, the gellan being contaminatedwith nucleic acid, thereby providing a mixture; and (b) maintaining themixture of step (a) under conditions where the DNase degrades at leastsome of the nucleic acid, thereby providing purified gellan. Optionally,a size-separation property modifying polymer such as poly(ethyleneoxide) may be added to the gellan or the purified gellan. In variousoptional embodiments, the gellan is contamined with more than 100 ppm,or more than 10 ppm of nucleic acid, based on weight parts of gellan.The method of the present invention can reduce the nucleic acidcontamination by 50% or more, e.g., to a level of less than 1 ppmnucleic acid based on weight parts of gellan. A DNase activating agentmay be added to speed to rate of nucleic acid degradation, where sodiumazide is a preferred DNase activating agent. Typically, the mixture ofstep (a) is maintained at about 30-45° C. for at least about 1 hour.Within less than about 24 hours, the nucleic acid has been essentiallycompletely degraded. After the gellan has been treated to degrade thenucleic acid, the DNA may, optionally, be deactivated. For example, thetreated mixture may be taken to a DNase inactivating temperature inexcess of about 50° C.

The present invention thus provides gellan having very low levels ofnucleic acid contamination. For example, the gellan may be in mixturewith either no nucleic acid, or nucleic acid at a concentration of lessthan 10 ppm, or less than 5 ppm, or less than 1 ppm nucleic acid, wherethe ppm values are based on weight parts of gellan. These purifiedgellans are particularly useful in preparing an electrophoresis medium.For instance, the purified gellan may be in combination with a buffercomposition suitable for maintaining said composition at a pH of 5-9. Abuffer composition with imidazole or a salt thereof and boric acid or asalt thereof is a preferred buffer composition, where the buffer mayadditionally contain EDTA or a salt thereof. To form a suitable gel forelectrophoresis, the purified gellan is in combination with across-linking agent. A preferred cross-linking agent is cystamine.

The present invention provides a superior method for treating gellan.Alternative methods require that the preparations be diluted, run overcolumns and then reprecipitated in an organic solvent. This is laborintensive and expensive. Our easy method allows gellan preparations tobe used as an economical electrophoresis matrix that can be used as anagarose alternative.

The present invention also provides kits useful in gel electrophoresis.In one aspect, the invention provides a kit that contains: (a) a matrixcomposition comprising gellan and nucleic acid at a concentration ofless than 10 ppm based on the weight of the gellan; (b) buffer; and (c)cross linking agent such as cystamine. The kit may optionally contain asize-separation property modifying polymer such as poly(ethylene oxide).The gel matrix may optionally include boric acid or a salt thereofand/or imidazole or a salt thereof, and may be at a pH between about 6.5and 8.5. The kit may also contain a separate container of buffer, e.g.,imidazole and boric acid buffer. The matrix may optionally include a DNAstain.

The invention also provides a method of performing electrophoresis. Themethod includes forming an electrophoresis medium by combiningingredients that include: (a) a matrix composition that includes gellan,nucleic acid at a concentration of less than 10 ppm based on the weightof the gellan, and size-separation property modifying polymer; (b)buffer; and (c) cross linking agent. The electrophoresis medium isnormally placed in an electrophoresis chamber and an electric field isapplied across the medium.

The invention also provides an electrophoresis apparatus. The apparatusincludes: (a) a cross linked matrix formed by combining gellan incombination with nucleic acid at a concentration of less than 10 ppm (orless than 5 ppm, or less than 1 ppm) based on the weight of the gellan(preferably prepared according to the methods of the present invention),cross linking agent, buffer, and size-separation property modifyingpolymer; and (b) an apparatus for exposing said cross linked matrix toan electric field.

The present invention also provides a method for recovering a biologicalmaterial. This method includes: (a) adding a mixture comprising abiological material to a cross linked electrophoresis medium, the mediumbeing formed by a method comprising combining a cross linking agent andgellan contaminated with less than 10 ppm (or less than 5 ppm, or lessthan 1 ppm) nucleic acid based on the weight of the gellan (preferablyprepared according to the methods of the present invention); (b)exposing the medium to an electric field to separate in said medium saidbiological material from other components in the mixture; (c) removing azone of the medium containing the biological material from the medium;(d) exposing the removed zone to an agent that reverses the crosslinking of the medium, to provide liquefied electrophoresis medium; and(e) separating the biological material from the liquefiedelectrophoresis medium, thereby recovering the biological material. Thecross-linking agent may optionally be a divalent metal cation, where theagent that reverses the cross linking is a chelating agent.Alternatively, the cross-linking agent may be a diamine and the agentthat reverses the cross linking is pH modifying agent. Alternatively,the cross-linking agent has a disulfide bond, and the agent thatreverses the cross-linking is a reducing agent that can reduce adisulfide bond to two thiol groups.

The present invention also provides a composition that includes water,imidazole or a salt thereof, and boric acid or a salt thereof, wherethis composition is particularly useful as a buffer, particularly whenthe composition has a pH between 5 and 9, e.g., between 6 and 8. Toachieve this pH, the imidazole or salt thereof typically has aconcentration between 10 and 100 mM, e.g., 20-60 mM, or about 44 mM,while the boric acid or salt thereof typically has a concentrationbetween 50 and 500 mM, e.g., 100-300 mM, or about 200 mM. EDTA or a saltthereof may optionally be included in this composition

The buffering composition of the present invention can be used withcystamine as a cross-linking agent for gellan. Cystamine is a reversiblecross-linker that allows gellan preparations to form a gel. Theimidazole/boric acid buffering system of the present invention maintainsa pH range wherein the cystamine is kept in a protonated form and thusfunctions as a cross-linking agent. During gel electrophoresis, it isnot necessary to recirculate the imidazole/boric acid buffering systemof the present invention in order to retain its efficacy as a buffer,which is a great advantage of the present invention because bufferrecirculation is very inconvenient for researchers.

These and other aspects of the present invention are described ingreater detail below.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of describing the present invention, the following termshave the indicated meaning.

Definitions

The term “biomolecule” refers to nucleic acids such as DNA and RNA,oligonucleotides, peptides, proteins, and other biological materialsthat can be separated using electrophoresis techniques, includingmixtures thereof.

The terms “cross-linking agent” and “cross-linker” refer to an additivewhich induces or promotes the association of the gellan molecules insolution, resulting in gel formation. Controlled changes to the chemicalor physical structure of the cross-linking agent may optionally revertthe gel into liquid solution. Examples of cross-linking agents aredivalent cations and diamines, including diamines containing disulfidebonds.

The term “cystamine” refers to cysteine dimethyl ester. Cystaminecontains two amino groups and one disulfide bond, and may be used as across-linker for the gellan gel formation.

The terms “degrade” and “degradation” refer to depolymerization of anoligonucleotide or polynucleotide. Degradation of an oligonucleotide orpolynucleotide will generally occur through enzymatic hydrolysis ofinternucleotide phosphodiester bonds to release short oligonucleotidesand/or mononucleotides.

The term “divalent metal cation” refers to divalent group IIA cationssuch as Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, etc. and to divalent transition metalcations such as Zn²⁺, Mn²⁺, Cu²⁺, etc.

The term “diamine” refers to organic compounds having two amine groupssuch as ethylene diamine, and 1,3 diamino-2-hydroxypropane (DAHP), etc.The term “polyamine” refers to organic compounds having two or moreamine groups. A suitable polyamine is a star-shaped dendrite in whichthere are amino groups at the end of the arms of the star. Typically,the amine groups of a diamine or polyamine are separated from each otherby a hydrocarbon or hydrocarbon derivative chain.

The term “disulfide” refers to the —S—S— bond. An example of a compoundcontaining a disulfide bond is cystamine.

The term “DNase” refers to an enzyme that degrades DNA.

The term “DNase activating agent” refers to any material that may beadded to the gellan during the gellan purification process of thepresent invention, wherein the agent enhances the speed and/orcompleteness of the degradation of the nucleic acid contaminant ingellan as compared to the same process in the absence of such an agent.Agents that activate DNase activity are known in the art. Sodium azideis a DNase activating agent.

The terms “electroosmosis” and “electroosmotic flow” refer to themovement of a charged substance through a charged matrix or otherbarrier by way of an electric field-induced convective flow.

The term “electrophoretic mobility” refers to the steady-state velocityinduced per unit field strength for a selected biomolecule duringelectrophoresis. Electrophoretic mobility can be measured in terms ofthe time required for a biomolecule to travel a specific distance in thegel, or in terms of distance traveled by a molecular species from areference point along the length of the gel during a selected time.

The term “ethylene oxide” refers to a monomeric unit having the formula—CH₂CH₂—O—.

The terms “gellan gum” and “gellan” refer to a family of relatedcarbohydrate polymers produced by Sphinogomonas bacteria (previouslyidentified as Pseudomonas), and includes native gellan gum, clarifiedgellan gum, deacetylated gellan gum, gellan gum that is both clarifiedand deacetylated, chemically modified gellan, and gellan gum produced bygenetically engineered bacteria.

Native gellan is described in Kennedy, J. F., Carbohydrate Chemistry,page 630 (1988) Clarendon Press, Oxford, as an extracellular anionicpolysaccharide produced by the bacterium Pseudomonas eloclea (ATCC31461). According to Kennedy, gellan from this source is a partiallyO-acetylated linear polymer of D-glucose, L-rhamanose, and D-glucuronicacid, which has the basic repeating unit, excluding acetyl groups, of ?3)-β-D-Glcp-(1? ∝)-β-D-GlcpA-(1? 4) -β-D-Glcp-(1? 4)-a-L-Rhap-(1?, whichmay also be written as GlcA 1-4 Glu 1-4 Rha 1-3 Glu, where “GlcA”represents glucuronic acid, “Glu” represents glucose and “Rha”represents rhamanose. According to Kennedy, in gellan, 33% of themonosaccharide residues are oxidized monosaccharide residues, andcellobiuronic residues constitute 66 wt % of the monosaccharide residuesin the polysaccharide.

Gellan is also described in Aspingall (The Polysaccharides, vol. 2,Academic Press, 1983, page 479) as obtained from Pseudomonas elodea andcontains a glucose:rhamanose ratio of 2:1. Aspingall states that gellancould be obtained from Kelco, Division of Merck & Co., Inc. as PS-60.PS-60 is available in three grades, namely: (a) “native”, which contains11% uronic acid, 3% acetylated uronic acid, 10% protein, 7% ash, and a2:1 ratio of glucose to rhamanose; (b) “deacetylated”, which contains13% uronic acid, no acetylated uronic acid, 17% protein and 8% ash, witha 2:1 ratio of glucose to rhamanose; and (c) “deacetylated andclarified”, which contains 22% uronic acid, no acetylated uronic acid,2% protein, 9.5% ash, and a 2:1 ratio of glucose to rhamanose.“Clarified” gellan is described below.

Gellan is also described in the following references: U.S. Pat. Nos.4,326,052; 4,326,053; 4,377,636; and 4,385,123. Other descriptions ofgellan may be found in, for example, Jansson et al., Carbohydr. Res.124, 135, 1983; and Sanderson et al. Progress in Food and NutritionScience, vol. 7, (eds. G. O. Phillips, et al., p. 201, Pergamon Press,Oxford, 1984).

Certain gellans are currently commercially available. For example,GELRITE™ is produced from a naturally occurring polysaccharide afterdeacetylation and “clarification”, where clarification refers to aprocess wherein the polysaccharide is fully or partially removed fromthe bacterial debris. GELRITE™ is available from a variety of sourcesincluding, for example, Sigma Chemical Co., St. Louis, Mo. Essentiallythe same material is also available from Sigma Chemical under the tradename PHYTAGAR™.

The terms “nucleic acid” and “oligonucleotide” and “polynucleotide” areused interchangeably herein to refer to a polymeric form of nucleotides.In one aspect, the nucleic acid is at least five bases in length, i.e.,it contains at least five nucleotides. The nucleotides of the inventioncan be deoxyribonucleotides, ribonucleotides, or modified forms ofeither nucleotide.

The term “polypeptide” refers to a molecule including at least twolinked amino acid residues and derivatives thereof.

The term “poly(ethylene oxide)” refers to a molecule containing aplurality of ethylene oxide units. While a poly(ethylene oxide)necessarily contains a plurality of ethylene oxide groups, thepoly(ethylene oxide) referred to herein may, but not necessarily does,contain groups other than ethylene oxide groups. For example, thepoly(ethylene oxide) may contain terminal groups (i.e., groups at theends of the molecule) other than hydroxyl groups, e.g., hydrocarbongroups. As another example, the poly(ethylene oxide) may includepropylene oxide groups, i.e., groups of the formula —CH(CH₃)CH₂—O—. Thepoly(ethylene oxide) is preferably water-soluble. In one aspect of theinvention, ethylene oxide is the only repeating unit in thepoly(ethylene oxide), while in another aspect, at least 90 molar percentof the repeating units in the poly(ethylene oxide) are ethylene oxide.The number average molecular weight of the poly(ethylene oxide) usefulin gel electrophoresis is typically 100,000 to 5,000,000.

The term “purification” is defined as the DNase treatment of gellan thatdecreases, but does not necessarily eliminate, background fluorescencein the presence of DNA stains (e.g., ethidium bromide, SYBER Green, GelStar, etc.) where this background fluorescence is caused by interactionbetween high molecular weight DNA and the DNA stain. A “purified” gellanof the present invention has been subjected to DNase treatment such thatthere is less background fluorescence in the presence of DNA stainrelative to the amount of background fluorescence observed with thecorresponding non-purified gellan, i.e., the gellan that has not yetgone through the treatment of the present invention.

The term “reducing agent” refers to any agent that can affect thereduction of a disulfide bond, thereby breaking the bond without causinga chemical change on any other substituent on the cross-linking agent.An example of a reducing agent is dithiothreitol (DTT).

The term “reversibility” is used to refer to the ability of gellan gelsto be returned to a liquid state.

The term “size-separation property modifying polymer” refers to polymersthat can be incorporated into the gellan-containing gel of the presentinvention to alter the size-separation properties of the electrophoresismedium formed from the gellan. Examples of size-separation propertymodifying polymers include hydroxyethyl cellulose, dextran, ficoll,poly(alkyleneoxide) including polyethylene oxide, pullulan, starch, andlinear polyacrylamide.

The terms “zone” and “band” refer to a portion of an electrophoresismedium or gel that contains substantially one biological material.Depending on the purity desired in a particular application of thepresent invention, there may be some degree of other biomolecules in agiven zone in addition to the biological material that is to berecovered using the method of the present invention.

Description

The present invention is directed to a novel purified gellan and atreatment method that provides the purified gellan. Additionally, theinvention is directed to gellan compositions that are particularlyuseful as electrophoresis gels. The invention also provides apparatusand methods for performing high-resolution separation and recovery ofnucleic acids, proteins and other biomolecules. Furthermore, the presentinvention provides a buffer system that is particularly useful incombination with gellan.

As discussed above, gellan gum is a linear carbohydrate polymer producedby bacterial fermentation. See, e.g., in U.S. Pat. Nos. 4,326,052;4,377,636; 4,385,123 and European Patent No. 0 012 552, the entiredisclosure and contents of which are hereby incorporated by reference.The carbohydrate polymer consists of repeating tetrasaccharide unitscomposed of two glucose sugars, a rhamnose and a glucuronic acid. Thisstructure is described more completely in O'Neil et al., “Structure ofthe Acidic Polysaccharide by Pseudomonas elodato” in CarbohydrateResearch (1983), 124, 123-133 and Jansson et al, “Structural Studies ofGellan Gum, an Extracellular Polysaccharide Elaborated by Pseudomonaselodato” in Carbohydrate Research (1983), 124, 135-139. The gellan gumproduced by typical fermentation has both O-acetyl and O-L-glyceryl3-linked to glucose units. The acetyl groups can be removed duringprocessing and the resulting materials are called low acyl gellan gumsas described in Sanderson, Food Gels, P. Harris (ed.) Elsevier AppliedScience, (New York: 1990), 202-232. Commercially available low acylgellan gums are available under the tradenames KELCOGEL™, GELRITE™ andPHYTAGEL™ gelllan.

Gellan has a number of unique properties that make it a desirable mediumfor electrophoresis and a potential replacement for agarose as one ofthe most widely used electrophoresis gels. In the presence of across-linking agent, gellan gum forms strong gels in a range of polymerconcentrations and buffer compositions. Accordingly, these gels aresuitable for high-resolution electrophoresis and the subsequent recoveryof the separated biomolecules. Gellan-based gel has the additionaladvantage of being “reversible” in that the gel can be returned to aliquid state under relatively mild conditions, typically by sequesteringor chemically altering the cross-linking agent. Furthermore, gellanforms gel at substantially lower concentration than agarose.Electrophoresis gels having gellan contents of as low as 0.03 wt % maybe constructed, however the most useful range of gellan gels for DNAelectrophoresis is between 0.1 wt % and 0.5 wt %. In contrast, agarosegel formation typically requires the presence of 0.8-3 wt % agarose.Particularly when gel electrophoresis is followed by recovery of theseparated biomolecules, it is advantageous to use a minimum amount ofgellant (e.g., agarose, gellan) so that there is less gellant that needsto be separated from the recovered matrix.

A common step in electrophoretic separations is to add a fluorescent dyeto the gel matrix. The dye will preferentially bind to the biomoleculein the matrix. Thus, after the biomolecules have been resolved, theentire gel can be placed under a source of ultraviolet (UV) radiationand a prominent signal appears at positions in the gel where biomoleculeis located. The present inventors have discovered that when commercialgrade gellan is used as the gel matrix in electrophoresis separation,the expected bands due to fluorescent dye localization near biomoleculeare not observed as expected. Thus, this routinely used approach tolocating biomolecule-containing zones in electrophoretic separations wasfound to be unsatisfactory when commercial grade gellan was used as thegel matrix. The present inventors have not only identified the cause ofthis unexpected problem, but have also discovered a solution asdescribed herein.

The present inventors have discovered that gel cast from commerciallyavailable gellan gives off strong background fluorescence across thegel, where this background fluorescence partially, and sometimeslargely, obscures the fluorescence emitting from zones of separatednucleic acid molecules. The present invention provides a method forpurifying gellan based on the finding that commercial or other untreatedgellan contains nucleic acid contaminant associated with the gellan.Though the nucleic acid contaminant does not affect formation of the gelor the separation of the biomolecules during electrophoresis, itinterferes with the visualization of the biomolecules that have beenresolved, particularly when the biomolecules are nucleic acids.

Thus, the present invention provides a method for purifying gellancomprising the steps of (a) combining gellan and a DNase, where thegellan is contaminated with nucleic acid, thereby providing a mixture;and (b) maintaining the mixture of step (a) under conditions where theDNase degrades at least some of the nucleic acid, thereby providingpurified gellan. In various optional embodiments, the starting gellan iscontaminated with 1-1000 ppm nucleic acid based on weight parts ofpolysaccharide, or 10-1000 ppm nucleic acid, 1-500 ppm nucleic acid, or10-500 ppm nucleic acid, or 1-100 ppm nucleic acid, or 10-100 ppmnucleic acid, or 1-50 ppm nucleic acid, or 10-50 ppm nucleic acid. Incertain embodiments, the starting gellan is contaminated with at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 400, 450, 500, 600, 700, 800, 900, or 1000ppm nucleic acid based on weight parts of polysaccharide.

In contrast, commercially available gellan has about 20-30 ppm nucleicacid. For example, KELCOGEL® gellan has about 24 ppm nucleic acid,KELCOGEL F® gellan has about 20 ppm nucleic acid, and GELRITE® gellanhas about 28 ppm, where these values are obtained by measuring theabsorbance at 260 nm, i.e., the OD₂₆₀ value, as measured on aspectrophotometer using a 0.5 wt % aqueous gellan solution, andcalculating nucleic acid concentration using the Warburg-Christiancalculation method. The Warburg-Christian method is described in O.Warburg and W. Christian (1942) Biochem. Z. 310:384-424, and entailsmeasurement of absorbance ratios at 260 nm and 230 nm, and again at 260nm and 280 nm. Background correction is made using absorbance values at320 nm, taking into consideration the factors outlined by Warburg &Christian.

Gellan is commercially available in a solid form. In one aspect, solidgellan is combined with water to create a suspension of the gellan inwater. This suspension is conveniently created by combining about ca. 2or less grams of solid gellan with ca. 100 grams of water so as toprovide an aqueous suspension having about 2 wt % or less gellan. Inother words, 2 parts gellan are combined with 100 parts water. When theconcentration of gellan in the suspension is greater than about 2 wt %,then the suspension tends to be lumpy and non-uniform due to non-uniformwetting of the particulate gellan, where this consistency is lessamenable to action by DNase. The water that is used to prepare thegellan suspension is preferably of low ionic strength, such as deionizedor distilled water.

In order to expedite formation of the suspension, the gellan-watermixture is preferably maintained at slightly elevated temperature, e.g.,35-40° C., for approximately 2 hours with agitation. When highertemperature is used, the gellan tends to dissolve in the water ratherthan form a suspension in the water. While a solution of gellan may alsobe combined with DNase according to the present invention, it isobserved that DNase tends to degrade nucleic acid more slowly in agellan solution than in a gellan suspension. A gellan solution is alsosomewhat disadvantageous in that it tends to gel without the addition ofadded cross-linking agents, and thus becomes very viscous. In a gellansuspension, a gellan concentration of less than 2 wt %, e.g., 0.5 wt %,is generally preferred in order to create a mixture that has workableproperties. While temperatures of less than 37° C. may be used to formthe gellan suspension, it is observed that a uniform suspension tends toform more slowly as the temperature is reduced much below about 37° C.While agitation for about 2 hours is typically adequate to create auniform-appearing aqueous gellan suspension, either longer or shortertimes may be used. Longer agitation times tend to allow more of thegellan to dissolve in the water, with a concomitant increase inviscosity. Shorter agitation times do not always allow all of the gellanparticles to wet and become suspended in the water. In one aspect, themixture of gellan and water is maintained at 30-45° C. for at least 0.5hour, more typically at least 1 hour, and still more typically at least2 hours.

Maintaining the water and gellan mixture at about 37° C. is alsoconvenient because after the suspension has formed, it is at atemperature that is conducive for DNase activity. As defined herein,DNase is an enzyme that is capable of digesting or degrading nucleicacid, i.e., polynucleotide. Nucleases can be endo or exo. A commerciallyavailable and preferred DNase is known as DNase 1, which is anendonuclease. The DNase can simply be added to the aqueous gellansuspension to begin the nucleic acid degradation. At temperatures higherthan about 60° C., the DNase denatures and becomes inactive. Attemperatures lower than about 30° C., the DNA degrading activity of theDNase slows considerably. Accordingly, it is preferred but not necessarythat the gellan suspension or solution be maintained at about 37° C.during the time when the DNase is expected to degrade the nucleic acid.

The DNase may be added at any convenience concentration. In general, asmore DNase is added, the rate of nucleic acid degradation increases, butthe cost to prepare the purified gellan also increases due to theexpense of the DNase. A suitable concentration of DNase can be readilydetermined by using skill available to one of ordinary skill in the art.A method to monitor the nucleic acid degradation process is describedlater herein. A DNase concentration of about 1-10 units/mL in a 2 wt %gellan suspension is observed to provide a good rate for nucleic aciddegradation, i.e., most or all of the nucleic acid degrades within about6 hours, and the cost for the DNase is not excessive. In thisdescription, a unit is defined by the Kunitz assay, where one unitproduces an increase in absorbance of 1×10³/min under assay conditions.

In addition to the DNase, in one aspect of the invention a DNaseactivating agent is added to the water/gellan mixture to enhance thespeed of Dnase-induced degradation of the nucleic acid contaminant.DNase activating agents such as divalent cations are known in the art,however these also cross-link gellan. The present inventors unexpectedlyfound that sodium azide also appears to activate DNase 1. Withoutintending to be bound by their theory, the present inventors believethis is because their modification of the gellan polymer gives DNasebetter access to the contaminating DNA. The sodium azide also served toprevent bacterial growth during the incubation period. Sodium azide isinactivated by the heat treatment used to inactivate the Dnase asdiscussed below. As one means for adding the DNase activating agent tothe water/gellan mixture, it is convenient to prepare a solution of theDNase activating agent, and then add some of that solution to thewater/gellan/DNase mixture. For instance, sodium azide may be preparedas a 5 wt % solution in water, and then a sufficient amount of thissolution is added to the water/gellan/DNase mixture to provide a sodiumazide concentration of about 1-5 mM, e.g., 3 mM.

The extent of the degradation process can be monitored by taking smallsamples of the mixture and staining them with ethidium bromide followedby observing the fluorescence of the sample. An ethidium bromideconcentration of about 0.5 mM is suitable. The purification is at leastpartially complete when there is reduced background fluorescenceobserved in the sample. Another approach is to monitor the opticaldensity of a sample of gellan suspension, where the absorbance at 260 nmis directly proportional to the concentration of nucleic acid in thesuspension. For this procedure it is necessary to precipitate the samplewith iso-propanol or ethanol, then dry and resuspend the sample. Forexample, a 0.25 wt % gellan suspension in water, prior to addition ofDNase, may have an OD₂₆₀ of about 0.2, which corresponds to a nucleicacid concentration of about 0.3 μg DNA/mL solution, or 24 ppm nucleicacid based on weight parts of gellan. Over the course of about 2 hoursat 37° C. in the presence of 4 units DNase/mL solution, the OD₂₆₀ dropsto a level that corresponds to a nucleic acid concentration of less than0.05 μg DNA/mL solution, or in other words, less than 1 ppm nucleic acidbased on weight parts of gellan. This procedure is conveniently used fordetermining times and concentrations needed to optimize the nucleic aciddegradation reaction. It is not needed to prepare gellan for use in DNAelectrophoresis.

In general, any reduction in nucleic acid contamination of gellan isdesirable because this reduction means that there is less backgroundnoise created when the gellan is used in an electrophoresis gel, and anucleic acid intercalating agent is used to visualize the resolvednucleic acid. In one aspect of the invention, at least 50% of thenucleic acid initially present in contact with the starting gellan isdegraded by the DNase, as determined by the A₂₆₀ method described above.In other aspects, the present invention provides a method whereby thenucleic acid contamination is reduced to less than 60%, or less than70%, or less than 80%, or less than 90% of the starting nucleic acidcontamination, preferably to below 1 ppm nucleic acid based on theweight parts of gellan. Thus, in various aspects of the invention,purified gellan compositions are provided that comprise water andgellan, wherein (a) the composition contains either no nucleic acid ornucleic acid at a concentration of less than 10 ppm based on weightparts of gellan, or (b) the composition contains either no nucleic acidor nucleic acid at a concentration of less than 5 ppm, or (c) thecomposition contains either no nucleic acid or nucleic acid at aconcentration of less than 1 ppm.

Upon completion of the nucleic acid degradation, the DNase that is inmixture with the purified gellan may be deactivated by heating the DNaseto a deactivating temperature. This thermal deactivation may beaccomplished by, e.g., heating the aqueous DNase/gellan mixture to atemperature in excess of about 60° C. for a time in excess of about 1minute. After this thermal deactivation step, the purified gellan may becooled and store at ca. room temperature.

The thermal deactivation step may optionally be omitted, and the productsuspension provided to the end-user with active DNase. It is notnecessary to heat the purified gellan for the sole purpose ofdeactivating the DNase because in the normal course of gel preparation,the gellan solution will be heated to above 75° C. This heating step isperformed in order to, for example, completely dissolve the gellan inthe water and to provide a sufficiently low viscosity solution that itcan be easily poured into the mold that forms the electrophoresis gel.During this heating process, any active DNase present in the gellansuspension will typically be destroyed.

In one aspect of the invention, the purified gellan is used to form agel for electrophoretic separation of biomolecules. In the preparationof such a gel, there is no need to recover the purified gellan from thesuspension in order to use the gellan to form an electrophoresis medium.This is particularly true when the concentration of the gellan in thepurified gellan is greater than the concentration of gellan needed forthe gel. It is a simple matter to treat concentrated suspensions ofgellan and to dilute the purified gellan to a concentration suitable forforming a gel. In general, a typical gellan concentration for castingelectrophoresis gel is approximately 0.175 wt %. Accordingly, as amatter of convenience, the concentration of gellan in the purifiedgellan prepared according to the present invention should be greaterthan approximately 0.1 wt %. Thus, preparing an initial gellansuspension having a concentration of about 1.5-2 grams gellan/100 gwater, i.e., about 1.5-2 wt % is a preferred aspect of the presentinvention.

As mentioned above, the purified gellan gums of the present inventionmay be formed into electrophoresis gels. These gels may be formedaccording to techniques that are well known and commonly used in the artwhen, for example, agarose is the gel matrix. In general, gel slabs forgel electrophoresis can be prepared from aqueous gellan suspensionshaving gellan concentrations ranging from about 0.03 to 2 grams/100 gaqueous suspension, where a concentration range of 0.1 to 0.5 grams/100g aqueous suspension is preferred to impart a relatively high mechanicalstrength to the gel, without using more gellan than is necessary. One ofthe advantages to using gellan in gel electrophoresis is that a suitablegel slab can be prepared using less gellan than would be required if theslab were prepared from agarose. Thus, a concentrated gellan suspensionmay be diluted to about 0.3 g gellan/100 g water using, for example,additional water or buffer. The solution is then heated to ensure thatall gellan particles dissolve completely.

In preparing a gel for gel electrophoresis, the gellan gum may becross-linked to afford a gel having relatively high mechanical strength.High mechanical strength is generally desirable in a gel being used ingel electrophoresis. The cross-linking of gellan may be accomplished bytechniques known in the art, e.g., using either a divalent metal cationor a diamine, as two examples. When the cross-linking agent is adivalent metal cation, any of a variety of divalent metal cations can beused, such as group IIA metal cations, such as Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺,etc., or transition metal cations such as Zn²⁺, Mn²⁺, Cu²⁺, etc. Thedivalent metal cations are generally added to the gellan gum solution inthe form of a metal salt. Typically, the gellan gum is mixed with thedivalent metal cation cross-linking agent at a temperature above about60° C., and the resulting solution is allowed to cool to form the gel.This temperature will typically be sufficient to inactivate any activeDNase still present in the gellan suspension. The divalent metal cationis preferably also added to the electrophoresis buffer solution thatwill be used in conjunction with the electrophoresis medium, preferablyat a concentration of about 0.1 to 10 mmol/L.

In the case of divalent metal cation cross-linked gels, afterelectrophoresis, the separated bands can be detected using a variety ofmeans including specific stains or direct scanning of the gel. The bands(zones) containing the resolved solutes (biomolecules) can be recoveredby a variety of means including cutting the band out of theelectrophoresis gel, and combining the isolated band with a chelatingcompound specific for the divalent cation used as the cross-linkingagent. The chelating compound may be in solution form or attached to asolid substrate, such as an ion exchange resin.

Electrophoresis gels of the present invention employing diaminecross-linking agents can be formed in a similar manner as that followedwhen divalent cation is used as the cross-linker. Thus, the diamine maybe mixed with the gellan gum at a temperature above about 60° C. Anybuffer used in conjunction with the diamine cross-linked gels of thepresent invention should maintain the gel at a pH below the pK's of theamino groups of the diamine so that the amino groups are protonated whenthey are intended to function as cross-linking groups. The pH needs tobe selected in view of the selection of the diamine cross-linking agent.For example, the methyl esters (blocked carboxyl groups) of lysine,arginine, and histidine, all form stable gels at pH below 7, preferably5 to 7.

In the case of diamine cross-linked gels, after electrophoresis, theseparated bands can be detected using a variety of means includingspecific stains or direct scanning of the gel. The bands containing theresolved solutes (biomolecules) can be separated from the bulk matrix bymechanical means, e.g., a knife or spatula, and then the biomoleculepresent in the isolated band can be recovered by a variety of meansincluding adding a base to the gel either in solution form or attachedto a solid substrate, such as an ion exchange resin. Once the pH of theband is increased to the point where the amino groups of the diaminecross-linking agent are no longer largely protonated, the gel reverts toa liquid.

A preferred diamine for the present invention is cysteine dimethylester, also referred to as cystamine. Cystamine is formed from twocysteine methyl esters linked at the side chains through a disulfidebond, where each cysteine methyl ester has a blocked (methylated)carboxyl group and a free amino group. As a diamine, cystamine can beadded to a gellan solution as a cross-linking agent to form a strong andstable electrophoresis gel. The gel can be readily converted back tosolution in the presence of a reducing agent that breaks the disulfidelinkage between the two cysteine methyl esters. An example of a suitabledisulfide reducing agent is DTT (dithiothreitol).

Although the above-described gellan electrophoresis gel uses a diamine(cystine dimethyl ester) to form the gel, thiol groups can also beintroduced into the gellan gum polymer by covalent bonds. As mentionedabove, gellan gum has a charged carboxyl group that can bind to cations.The carboxyl groups can also be used as an attachment point to makevarious gellan gum derivatives. The carboxyl group is a reactive sitethat can be covalently attached to thiol or other functional groups. Thecarboxyl group reacts with amine-containing compounds optionally in thepresence of carbodimides. Carbodimides promote the condensation of anamine and a carboxyl group.

Alternatively, a derivative of gellan gum can be prepared that has freesulfide groups covalently attached to the carbohydrate chain. Forinstance, if gellan gum is reacted with an aminothiol compound such as2-mercaptoethylamine and a carbodiimide, the carboxyl group of thegellan and the amine group of the aminothiol compound form an amidebond. A gel-forming polymer having thiol groups has the advantage ofhaving no charge, and the free thiol groups may be used to formreversible gels based on the state of the solution.

In addition to using divalent metal cation or diamine (with or withoutdisulfide) cross-linking agents to modify the properties of the gellangels of the invention, the properties of these gels can be modified bythe incorporation of size-separation property modifying polymers intothe gels, as discussed next.

Gellan gum is an anionic polymer due to the presence of carboxylic acidgroups in the glucuronic acid residues in the polysaccharide backbone.The negative charge of the carboxyl group requires a counterion, andwhen an electric field is applied, these cationic counterions (alongwith waters of hydration) migrate to the negative electrode due toelectroosmosis. The result is a net flow of buffer toward the negativeelectrode. Negatively charged biomolecules such as DNA, which migrate tothe positive electrode during electrophoresis, are slowed in theirprogress as they have to journey against the opposing flow of thebuffer. Similarly, the negatively charged glucuronic acids in the gellanattempt to migrate to the positive electrode, thereby destabilizing thegel structure. Addition of certain water soluble polymers to the gellansolution prior to gel formation incorporates the water soluble polymersinto the electrophoresis gel. The presence of the water soluble polymeris observed to not only reduce electroosmosis, but also to increase theresolution of lower molecular weight biomolecules. These water solublepolymers, which are referred to herein as size-separation propertymodifying polymers, are described in Cole et al., “Modification of theElectrokinetic Properties of Reversible Electrophoresis Gels for theSeparation and Preparation of DNA”, Applied Biochemistry andBiotechnology, 82, 57-76, (1999).

A variety of polymers, both linear and branched, can be incorporatedinto the gellan gum electrophoresis gels to modify the size-separationproperties of the gel. Some examples of polymers which can be usedinclude, without limitation:

dextran, Ficoll, amylose, alginates, amylopection, xanthan gum, whelangum, hydroxethyl cellulose, methyl cellulose, poly(alkylene oxide) andparticularly poly(ethylene oxide), polyvinylpyrrolidone, andpolyvinylalchol. Typically, increasing the concentration of the polymerreduces the electroosmotic flow in the gels. It is also observed thathigher molecular weight polymers are typically more effective atreducing electroosmotic flow than are lower molecular weight polymers.

The gellan of the present invention may be combined with a buffersolution. Preferred buffer solutions are capable of maintaining the pHof the gellan-based electrophoresis gel within a range of about 5-9because some biomolecules are susceptible to damage at pH values outsidethis range. Suitable buffer solutions are selected based, in part, onthe type of cross-linking agent used during gel formation. For example,when the cross-linking agent is a diamine, it is desired that the buffersolution maintain the pH of the gel below the pKa of the diamine toensure that the amino groups are protonated. Examples of bufferssuitable for the gellan-based electrophoresis gels of the presentinvention are shown in the Table below.

TABLE Buffers Used for Gellan Gum Gel Electrophoresis Buffer CompositionpH TB 0.045 mol/L tris(hydroxymethyl)aminomethane (Tris) and 8.5 0.045mol/L boric acid TA 0.045 mol/L tris(hydroxymethyl)aminomethane (Tris)8.3 and 0.045 mol/L boric acid TG  0.04 mol/L TRIS and 0.1 mol/L aceticacid 8.3 BBE 0.022 mol/L bis (2-hydroxethyl) imino-tris (hydroxymethyl)6.8 methane, 0.045 mol/L boric acidDuring electrophoresis, positively charged ions in the buffer move tothe negative electrode, while negatively charged ions move to thepositive electrode. Due to electroosmosis of the buffer duringelectrophoresis, a pH gradient may form across the gel between thepositive and negative electrodes. This is problematic because pHvariation may cause the diamine cross-linker to become deprotonated,thereby adversely affecting the gel strength. It can also cause heatbuild-up. Typically, a buffer solution needs to be rapidly recirculatedduring electrophoresis in order to eliminate this pH gradient. However,a buffer solution with strong buffering capacity is able to maintain thedesired pH without recirculation. The present invention provides abuffer composition having strong buffering capacity comprising imidazoleand boric acid, which is capable of maintaining the pH within the rangeof 6.5-8.5. EDTA may optionally be added to the buffer solution. Anexample of a buffer composition of the present invention is 20-60 mM(e.g., 44 mM) imidazole and 100-300 mM (e.g., 200 mM) boric acid, whichis capable of maintaining pH at 6.5-8.5 without being recirculatedduring electrophoresis. 1-3 mM (e.g., 2 mM) of EDTA may be optionallyadded to this buffer composition.

The present invention further provides a kit useful in preparing anelectrophoresis gel. A kit of the invention includes gellan, preferablyin suspension or solution form, a buffer and a cross-linking agent. Boththe gellan solution/suspension and the buffer can be present inconcentrated form and then diluted to the proper concentration forgel-casting. In one embodiment, each of the kit components is inseparate containers. However, the kit may alternatively, oradditionally, contain purified gellan in pre-cast form, i.e., the gellanis in combination with, and has been cross-linked by, the cross-linkingagent and has been poured into a form that is ready to place directlyinto an electrophoresis device. When the kit contains gellan only inpre-cast form, the kit may or may not contain cross-linking agent in aseparate container. The buffer is the running buffer for the gelelectrophoresis, and may optionally be in combination with the gellan sothat the gellan is provided in solution or suspension form. Depending onthe biomolecules to be resolved, the gellan solution/suspension mayoptionally contain a size-separation property modifying polymer asdescribed above. In addition, dye may optionally be added to the gellansolution/suspension to help with visualizing the wells for gel loading.Examples of suitable dyes include bromophenol blue, xylene cylanole, andorange G. Likewise, nucleic acid stain, such as ethidium bromide orSyber green may also be added to the gellan solution/suspension, aloneor in combination with the dye, depending on the biomolecules to beresolved. The concentration of these materials that may be included inthe gellan gel are the same concentrations as are currently used in theart when these materials are utilized in electrophoresis gels made fromalternative materials. For example, a concentration of ethidium bromideof about 0.5 mM is suitable in many instances.

In another aspect, the present invention provides a method of performingelectrophoresis that includes forming an electrophoresis gel medium bycombining ingredients that include (a) a matrix composition comprisinggellan solution, nucleic acid at a concentration of less than 10 ppmbased on the weight of the gellan, and a size-separation propertymodifying polymer; (b) a buffer; and (c) a cross-linking agent.

The present invention further provides an electrophoresis apparatus thatincludes (a) a cross-linked matrix formed by combining gellan solution,cross-linking agent, nucleic acid at a concentration of less than 10 ppmbased on the weight of the gellan, buffer, and size-separation propertymodifying polymer; and (b) an apparatus for exposing said cross-linkedmatrix to an electric field, including a variety of conventional formatssuch as flat bed apparatus, vertical slab apparatus, tubes and capillarytubes.

In addition, the present invention provides a method for recovering abiological material, where the method includes (a) adding a mixture thatincludes a biological material to a cross-linked electrophoresis medium,the medium being formed by a method that includes combining across-linking agent and gellan contaminated with less than 10 ppmnucleic acid based on the weight of the gellan; (b) exposing the mediumto an electric field to separate in said medium the biological materialfrom other components in the mixture; (c) removing a zone of the mediumcontaining the biological material from the medium; and (d) exposing theremoved zone to an agent that reverses the cross-linking of the mediumto thereby provide liquefied electrophoresis medium. Optionally, themethod further includes a step (e) separating the biological materialfrom the liquefied electrophoresis medium, thereby recovering thebiological material; or a step (f) using the solution without removingthe polymer.

Electrophoresis gels made from the purified gellan of the presentinvention afford many desirable properties and advantages overalternative gel matrix compositions. One primary advantage is that thepurified gellan of the present invention can be formed into a gel matrixfor electrophoresis, where that gel matrix emits less backgroundfluorescence in those instances where nucleic acid is separated on thegel matrix and a fluorescent intercalating agent is used to visualizethe bands of resolved nucleic acid. In addition, the gel matrix madefrom the purified gellan is non-toxic, however it may optionally containsome DNA intercalating agent, e.g., ethidium bromide. However, thepresent inventors recommend using a non-toxic stain like Syber Green,which actually improves the visibility of the bands. The gellan matrixis very easy to cast, in that one needs only to heat the purified gellansolution to a temperature of 75° C. or greater, add the cross-linker,and then pour the matrix into the electrophoresis mold. In contrast,many agarose-based products require a step of measuring variouscomponents, mixing them together, and heating to 100° C., whichincreases the risk of burning the user and sometimes causes the solutionto boil over the top of the flask. Thus, an electrophoresis gel madefrom the prepared gellan matrix is much easier, safer, and quicker toprepare than a corresponding gel made from, e.g., agarose.

Also, in contrast to many agarose-based products, the gellan gels of thepresent invention provide better commercial value in that relativelyless of the gellan is required to make a gel having the desiredconsistency for gel electrophoresis. Additionally, a gel matrix madefrom the purified gellan of the invention affords really sharp bandswhen linear DNA fragments are separated, and the resolving power of thegel matrix is also very good. An added advantage is that more DNA can beloaded on the gel without sacrificing band sharpness and resolution.

The gellan gels readily set up into a hard matrix upon cooling to about30° C., and can be re-liquefied by heating to about 95°-100° C.,depending upon other gel components that are present. The initialmelting of the purified gellan prior to forming the electrophoresis gelmatrix, as well as an optional re-heating to re-liquefy the gel matrixin the event it undesirably solidifies, can be readily achieved bysimply placing the composition into a microwave oven and bringing to theboiling point.

Alternatively, the product matrix can be returned to a liquefied formupon breaking the cross-linking bonds, where this bond breaking processis initiated under conditions specific to the cross-linker, e.g., usinga reducing agent when the cross-linker contains a disulfide bond, orraising the pH when the cross-linker contains protonated amines, oradding a chelating agent when the cross-linker is a divalent metal ion.In effect, this allows the gellan gels of the present invention to beliquefied by breaking the bonds chemically rather than melting the gelat high temperatures, offering significant advantages over existingelectrophoresis gels. Currently, almost all DNA is extracted by meltingthe agarose gels, which are typically “low-melting point agarose” gelsthat melt at a lower temperature than regular agarose and offer less ofa chance for heat damage to occur to the DNA sample. Despite thisimprovement, heat damage can still occur because most low melting pointgels require temperatures of 55° C. to 65° C. in order to actually melt,and the proteins and the DNA components begin to denature at 55° C. Incontrast, the gellan gels of the present invention provide a higherquality DNA sample because they are not subjected to temperatures thatcan damage the DNA. Low-melting point agarose is also problematicbecause it cools quickly and often solidifies in equipment (e.g.,pipettes), often requiring time-consuming recovery procedures or theuser to start over again. This is not a problem with the gellan-basedgels of the present invention because they remain liquid at roomtemperature once reversed chemically.

In summary, the gellan-based gels of the present invention are easier touse, save time, and produce a higher quality DNA sample than existingpreparative gels. In addition to the foregoing desirable propertiesand/or advantages of the gellan-based gels of the present invention, ithas been observed that persons working with the biomolecules resolvedusing the gellan-based gels can manipulate those resolved biomoleculeswhen those molecules are still in combination with the gel matrix. Thus,certain reactions can be conducted on the excised resolvedbiomolecule(s), and it is not necessary to purify the biomolecule fromthe gellan.

For instance, nucleic acids, such as DNA, are examples of biologicalmaterial that can be recovered through electrophoresis usinggellan-based gel. After separation, bands containing DNA can be excisedwith a blunt spatula and placed in a microfuge tube. In the case of DNAisolated from gels cast with divalent cation such as Ca²⁺, aconcentrated solution of 0.5 M EDTA (pH 8.0) may be added to the gelslice so the final EDTA concentration is about 5 mM. In the case of DNAisolated from gels cast with a diamine (e.g., DAHP), a concentratedsolution (pH 8.5) of TRIS may be added to the gel slice so the finalconcentration is 50 mmol/L TRIS. In the case of DNA isolated from gelscast with cystamine, a concentrated solution of DTT may be added to thegel slice so the final concentration of DTT is about 10 mmol/L. Gentlemixing is typically sufficient to dissolve the gel. The reaction isfaster if the gel slice and DTT are incubated at 45 to 55° C.Alternatively, DNA can be gently and efficiently recovered from gellangels that are cross-linked cystamine by means of a gellan-digestingenzyme, gellanase (U.S. Pat. No. 5,342,773).

DNA isolated from gellan electrophoresis gel can be readily cut by avariety of restriction enzymes (e.g., Eco RI, Hind III, and Bam H1) inthe presence of gellan gum. The restriction enzyme (ca. 10 units) can beadded directly to the dissolved gel band and the solution mixed. A 10×restriction buffer should then be added, and the tube contents mixed andincubated at 37° C. for 2-4 hrs. The restriction fragments can beanalyzed by electrophoresis.

The activity of DNA ligase is not significantly inhibited by gellan gumas determined by analysis of the restriction fragments using agarose gelelectrophoresis. Ligase (1 unit) can be added directly to a mixture ofthe restriction fragments resulting from the restriction digestion asmentioned above. 5× ligase buffer may then be added, the tube contentsmixed together and incubated at 37° C. for 2-4 hrs. Successful ligationof the DNA restriction fragments is indicated by the formation of highermolecular weight products when analyzed by gel electrophoresis.

DNA isolated from gellan gum electrophoresis gels can be used in directtransformation of E. coli. The dissolved gel solution (e.g., 0.05 mL)containing isolated DNA may be placed on ice and competent cells (e.g.,0.05 mL) are added to a final volume of ca. 0.1 mL. Transformation canbe done according to the supplier's instructions. Typically, thisconsists of incubation on ice for 30 min, heat shock at 37° C. for 45seconds, cooling on ice for 2 min, and addition of 0.95 mL of LB media.The tubes are then incubated at 37° C. with shaking (225 rpm) for about1 hr. The cells are diluted and plated out on LB plates containingantibiotics. Colonies are counted the next day after incubation at 37°C.

DNA isolated from gellan gum electrophoresis gels followed by ligationto other DNA transforms E. coli competent cells with about the samefrequency as low melting point agarose. To remove gellan from adissolved gellan gel sample containing isolated target biomolecule, suchas DNA, a solution of CaCl₂ may be added to the sample to aconcentration of ca. 5 mmol/L or greater. The solution is mixed and thecross-linked gellan gum can be removed by centrifugation, for example at12,000×g for 15 min, or by filtration. The gellan gum is collapsed intoa compact pellet (centrifugation) or retained on a filter (filtration)leaving the target molecule in solution. Alternatively, the gellan slicecan also be soaked for a short while in a diffusion buffer [0.5MAmmonium acetate, 1 mM EDTA] at 37° C. The gel is then centrifuged asabove. The solution on top is then brought to 2.5 M ammonium acetate andthe DNA precipitated with 2½ volumes of ETOH. This diffusion buffer maybe a component in kit of the present invention.

Proteins can also be separated by electrophoresis using gellan gel. andpositively charged proteins move towards the negative electrode. Addingsize-separation property modifying polymers to the gel can reduce theelectroosmotic flow contributed by the charge on the gellan matrix. Theyalso aid in separation.

The following examples are supplied so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention.

EXAMPLES Example 1 Gellan Purification

A solution of 35 mM sodium azide and 5 μ/ml DNase 1 in deionized waterwas made, to which commercial gellan [Kelcogel Fine (KGF), purchasedfrom CPKELCO] was added to make a 1.75 wt % gellan suspension. This is10 times more concentrated than the final gellan solution ready forgel-casting. The mixture was shaken at 37° C. At any point, 10 ml of thesuspension could be extracted and diluted with 1 ml of buffer. 0.5 mMethidium bromide could be added before the mixture is placed on a UVtransilluminator to monitor the loss of fluorescence, which indicatesthe degree of nucleic acid degradation. Typically, there was nodetectable nucleic acid contaminant after the suspension had been shakenfor 20 hours. The mixture was then heated to 75° C. for 4 hrs todeactivate the DNase 1. The resulted gellan solution can be useddirectly or diluted for casting electrophoresis gel. The purified gellanmay be isolated from the solution, however, it may also be retained inthe solution and an electrophoresis slab formed from the solution.

Example 2 Buffer Preparation

A mixture of 0.44 M imidazole, 2.0 M boric acid and 0.02 M EDTA indeionized water was stirred at room temperature for 2-3 hrs until allthe components were dissolved. Optionally, 5 μg/ml ethidium bromidecould be added. The resulting buffer is 10 times more concentrated thanwill typically be used in gel electrophoresis. It should be diluted foruse in gel-casting and performing the electrophoresis.

Example 3 Size-seperation Modifying Polymer Solution Preparation

While the size-separation modifying polymers are all water-soluble,somewhat different procedures may be followed in order to dissolve themin solution forming polymer solutions may require different caredepending on their molecular weight (MW).

For example, poly(ethyleneoxide) (MW=4,000,000) was added to deionizedwater to make a 0.5 wt % mixture. It was necessary to agitate themixture for about 24 hours to ensure that all the polymer particles hadcompletely dissolved.

Poly(Poly(ethyleneoxide) (MW=100,000) was added to deionized water tomake a 2 wt % mixture. The mixture was heated to boiling to dissolve allthe polymer particles.

Example 4 General Procedure for Gel Formation and Electrophoresis

The concentrated gellan solution prepared according to Example 1 wasmixed with the buffer solution of Example 2 and diluted with deionizedwater so the final concentrations are: gellan at 0.17 wt %, imidazole at44 mM and boric acid at 200 mM. Optionally the solution could be furthercombined with the size-separation property modifying polymer solution,where the final concentration of the polymer can be adjusted dependingupon the biomolecules to be resolved. The solution was then heated to atemperature in excess of 75° C. At this point, cystamine was added sothat its final concentration was between 2.5 to 10 mM. Optionally,nucleic acid stain may also be added. The solution was then poured intothe gel tray and allowed to solidify. A comb was then suspended in thegel to form the sample wells. A flat bed submarine gel electrophoresisapparatus was used. The electrode buffer chambers were filled with theelectrophoresis buffer (44 mM imidazole and 200 mM boric acid). Thesamples of biomolecules were diluted with a buffer solution containing atrace of bromophenol blue, so the final concentration of the samples wasapproximately 2 wt %. The samples were loaded into the wells. 2.5 mMcross-linker was added to the anode chamber and the electric field wasapplied. Typically, the gel can be run at 7 volts/cm.

Example 5 Formation of Gellan Electrophoresis Gels Using Cystamine andGel Liquefaction

Strong stable gels were formed when cystamine (5 mM) in imidazole/boricacid buffer (44 mM imidazole, 200 mM boric acid, pH=6.8) was added togellan suspension to form a gellan electrophoresis gel (0.1 wt %). Asolution of dithiothreitol (0.01 mol/L) was added to the gellanelectrophoresis gel (0.01%) whereupon the gel converted back tosolution.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A purified gellan composition containing nucleic acid at aconcentration of less than 10 ppm based on the weight of gellan preparedby combining DNase and an unpurified gellan composition contaminatedwith nucleic acid to make a mixture wherein said mixture is maintainedunder conditions such that DNase degrades at least some of the nucleicacid, thereby resulting in the purified gellan composition.
 2. Thepurified gellan composition of claim 1 wherein the concentration ofnucleic acid in the unpurified gellan composition is more than 100 ppmbased on the weight of gellan.
 3. The purified gellan composition ofclaim 1 wherein the purified gellan composition contains less than 50%of the nucleic acid in the unpurified gellan composition.
 4. Thepurified gellan composition of claim 1 wherein said mixture furthercomprises a DNase activating agent.
 5. The purified gellan compositionof claim 4 wherein the DNase activating agent is sodium azide.
 6. Thepurified gellan composition of claim 1 wherein said mixture ismaintained at 30-45° C. for at least 1 hour.
 7. The purified gellancomposition of claim 1 wherein in preparing the purified gellancomposition, nucleic acid degradation is monitored.
 8. The purifiedgellan composition of claim 1 wherein after the mixture is maintainedunder conditions such that DNase degrades at least some of the nucleicacid, the DNase is deactivated.
 9. The purified gellan composition ofclaim 8 wherein the DNase is heat deactivated by heating the DNase to aninactivating temperature in excess of 50° C.
 10. The purified gellancomposition of claim 1 wherein the DNase is DNase
 1. 11. The purifiedgellan composition of claim 1 wherein in preparing the purified gellancomposition, boric acid is added to the unpurified gellan composition orthe purified gellan composition.
 12. The purified gellan composition ofclaim 1 wherein in preparing the purified gellan composition, imidazoleis added to the unpurified gellan composition or the purified gellancomposition.
 13. The purified gellan composition of claim 1 wherein inpreparing the purified gellan composition, a size-separation propertymodifying polymer is added to the unpurified gellan composition or thepurified gellan composition.
 14. The purified gellan composition ofclaim 13 wherein the size-separation property modifying polymer ispoly(ethylene oxide).
 15. A gellan composition comprising water andgellan, the composition containing either no nucleic acid or nucleicacid at a concentration of less than 10 ppm based on the weight of thegellan.
 16. The gellan composition of claim 15 that contains either nonucleic acid or nucleic acid at a concentration of less than 5 ppm basedon the weight of the gellan.
 17. The gellan composition of claim 15 thatcontains either no nucleic acid or nucleic acid at a concentration ofless than 1 ppm based on the weight of the gellan.
 18. A gellancomposition, comprising: (a) gellan; and (b) either no nucleic acid ornucleic acid at a concentration of less than 10 ppm nucleic acid, basedon the weight of gellan.
 19. The composition of claim 18 furthercomprising a size-separation property modifying polymer.
 20. Thecomposition of claim 19 wherein the size-separation property modifyingpolymer is poly(ethylene oxide).
 21. The composition of claim 18 furthercomprising a buffer for maintaining said composition at a pH of 5-9. 22.The composition of claim 21 wherein the buffer comprises imidazole or asalt thereof and boric acid or a salt thereof.
 23. The composition ofclaim 18 further comprising EDTA or a salt thereof.
 24. The compositionof claim 18 further comprising a size-separation property modifyingpolymer, imidazole or a salt thereof, boric acid or a salt thereof, andEDTA or a salt thereof.
 25. The composition of claim 18 furthercomprising a cross-linking agent.
 26. The composition of claim 25wherein the cross-linking agent is cystamine.
 27. A kit comprising: (a)a matrix composition comprising gellan and nucleic acid at aconcentration of less than 10 ppm based on the weight of the gellan; (b)buffer; and (c) cross linking agent.
 28. The kit of claim 27 wherein thenucleic acid is present in the matrix composition at a concentration ofless than 5 ppm based on the weight of the gellan.
 29. The kit of claim27 wherein the matrix composition further comprises a size-separationproperty modifying polymer.
 30. The kit of claim 29 wherein thesize-separation property modifying polymer is poly(ethylene oxide). 31.The kit of claim 27 further comprising a size-separation propertymodifying polymer.
 32. The kit of claim 31 wherein the size-separationproperty modifying polymer is poly(alkylene oxide).
 33. The kit of claim27 wherein the matrix composition further comprises boric acid or a saltthereof.
 34. The kit of claim 27 wherein the matrix composition furthercomprises imidazole or a salt thereof.
 35. The kit of claim 27 whereinthe matrix composition has a pH between 6.5 and 8.5.
 36. The kit ofclaim 27 wherein the matrix composition further comprises a DNA stain.37. The kit of claim 27 wherein the buffer comprises imidazole or a saltthereof.
 38. The kit of claim 27 wherein the buffer comprises boric acidor a salt thereof.
 39. The kit of claim 27 wherein the buffer comprisesimidazole or a salt thereof, and boric acid or a salt thereof.
 40. Thekit of claim 27 wherein the cross linking agent is cystamine.